Struktur und Funktion von Transaminasen aus Corynebacterium glutamicum

Marienhagen, Jan

Dissertation / PhD Thesis/Book

Struktur und Funktion von Transaminasen aus Corynebacterium glutamicum

Marienhagen, J. (Corresponding author)FZJ*

2008
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 978-3-89336-512-8

Jülich : Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag, Schriften des Forschungszentrums Jülich. Reihe Gesundheit / Health 4, VI, 154 S. (2008) = Universität Düsseldorf, Diss., 2007

Please use a persistent id in citations: http://hdl.handle.net/2128/3094

Abstract: In the pathways of most amino acids transaminases reversibly catalyze the transfer of an amino group from an $\alpha$-amino acid to an $\alpha$-oxo-acid. Such enzymes are characterized by great sequence homology, similar structures and overlapping substrate specificities. The main objective of this thesis was to gain detailed knowledge of the structure and function of transaminases of Corynebacterium glutamicum with an emphasis on the enzymes participating in the synthesis of the three branched chain amino acids. Twenty proteins containing a transaminase motif were isolated and characterized concerning their substrate spectrum. The in vivo function was studied by chromosomal deletion mutants. Those experiments lead to the identification of the alanine transaminase AlaT and the aspartate transaminase AspC, which is important for lysinesynthesis. Two proteins could be assigned as cysteine desulfurases, which are involved in the assembly of FeS-clusters. The transaminase IlvE for the synthesis of the three branched chain amino acids catalyzes the formation of L-leucine, L-isoleucine and L-valine with comparable specific activities of 9.6 – 13.9 $\mu$mol min$^{-1}$ mg (protein)$^{-1}$. However, in vivo this enzyme is only essential for the L-leucine- and L-isoleucine-synthesis. The alanine-valine transaminase AvtA, which is strictly alanine-dependent, contributes also to the synthesis of the three branched chain amino acids and catalyzes the formation of valine with a high specific activity of 18.2 $\mu$mol min$^{-1}$ mg (protein)$^{-1}$. This activity alone ensures valine-synthesis in an ilvE-deletion mutant. The transaminase AroT is involved in the formation of L-phenyalanine and L-tyrosine. Interestingly, this enzymes catalyzes the conversion of 2-oxo-iso-caproate to L-leucine with less than 10 % of the specific activity of L-phenylalanine-synthesis from phenylpyruvate, but is unable to transaminate the precursors of L-isoleucine or Lvaline. Directed evolution generated an AroT-mutein with a M54L-mutation, which converts 2-oxo-iso-caproate to L-leucine with an almost doubled catalytic efficiency of 59 M$^{-1}$ s$^{-1}$. The histidinol-phosphate transaminase HisC was crystallized and a structure model up to a resolution of 1.8 A was calculated. The model included 364 of 366 amino acids and gave information about the amino acid residues participating in the catalysis of this enzyme. The introduction of active-site mutations almost always resulted in a decreased activity of HisC, but a Y123F-mutation doubled the specific activity for the transamination of 4-hydroxy-phenylpyruvate to L-tyrosine (0.5 to 0.9 $\mu$mol min$^{-1}$ mg (protein)$^{-1}$). L-alanine accumulates during the microbial production of L-valine with C. glutamicum. Single deletion of the genes for the alanine-valine transaminase (avtA) and the alaninetransaminase (alaT) in the genome of a L-valine-production strain decreased the formation of this byproduct and increased the synthesis of L-valine. During a batchfermentation the loss of AlaT-activity reduced the L-alanine concentration in the culture filtrate by 75 %.

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